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Genomics and Epigenetics

Get Prepped: Nanopore Library Preparation Optimization

Nanopore is a relatively new sequencing platform and researchers are still trying to optimize the protocol for their own specific applications. In our lab, we work primarily with metagenomic samples and use the 1D sequencing kits. Over the past year, we have optimized this technique. To check the quality of the Nanopore library preparation we…

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CRISPR-Based Activation (CRISPRa) of Genes: A How-To Guide

The purpose of this article is to walk you through the steps needed to overexpress genes using CRISPR/Cas9-based activation (CRISPRa). A broader overview of this topic (including CRISPR-based repression) can be found here. CRISPR/Cas9 is more than a programmable nuclease. When stripped of its nuclease activity, it can activate and repress transcription, alter chromatin structure,…

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CRISPR-Cas9 Genome Editing: Weighing the Pros and Cons

Genome editing is a hugely powerful tool which can help you to address a multitude of questions in your research. However, it is not necessarily the best tool for the job in every situation. Below is a discussion of the main advantages and disadvantages associated using CRISPR-Cas9 for genome editing. The Pros It’s Simple to…

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Use ddRAD-seq to Study Non-Model Organisms

Reduced-representation genome sequencing has been one of the most important advances in the last several years for enabling massively parallel genotyping of organisms for which there is no reference-grade genome assembly. An implementation of the approach known as ddRAD-seq, first conceived in the Hoekstra lab at Harvard, has been widely adopted by the plant and…

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Reducing GC Bias in WGS: Moving Beyond PCR

WGS technologies have seen significant progress since the completion of the Human Genome Project in 2003. First-generation Sanger Sequencers were limited by lengthy run times, high expenses, and throughputs that read only tens of kilobases per run. The arrival of second-generation sequencers in the mid-2000s brought about the plummeting of sequencing costs and run times,…

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How to Improve Your WGS DNA Library

In whole genome sequencing (WGS) initiatives it is not enough to simply sequence the whole length of the genomic DNA sample just once. This is because genomes are usually very large. The human genome, for example, contains approximately 3 billion base pairs. Although sequencing accuracy for individual bases is very high, when you consider large…

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Generating RNA-seq Libraries from RNA

One of the most powerful methods of modern cellular biology is creating and analyzing RNA libraries via RNA-sequencing (RNA-seq). This technique, also called whole transcriptome shotgun sequencing, gives you a snapshot of the transcriptome in question, and can be used to examine alternatively spliced transcripts, post-transcriptional modifications, and changes in gene expression, amongst other applications.…

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Battle of the Methods: Whole Transcriptome Versus mRNA-seq

Maybe you want to examine the entire transcriptome or maybe you want to investigate changes in expression from your favorite gene. You could do whole transcriptome sequencing or mRNA-seq. But which one is right for your project? From budget considerations to sample collection, let’s briefly look at both to see which might be best for your…

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Analyzing RNA-Seq Data

RNA-seq is based on next-generation sequencing (NGS) and allows for discovery, quantitation and profiling of RNA. The technique is quickly taking over a slightly older method of RNA microarrays to get a more complete picture of gene expression in a cell. Data generated by RNA-seq can illustrate variations in gene expression, identify single nucleotide polymorphisms…

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Kick-Start Your Gut Microbiome Study in Four Easy Steps

Today, the gut microbiome is garnering a large amount of media attention for its role in human health and disease. From influencing immune responses to impact our brain, the gut microbiome is an important and necessary aspect of our life. So much so, that current investigations in the gut microbiome are focusing on developing biomarkers for…

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Studying the Epigenome by Next Generation Sequencing

The epigenome has been in the research spotlight, and for good reason. Not only has it been associated with the developmental stages of an organism, but epigenetic alterations lead to disorders and have been linked to many human diseases. So, the question stands: what exactly is an epigenome? What Is the Epigenome? Simply put, the…

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Using dbSNP and ClinVar to Classify Gene Variants

As we discussed previously, the gaps in our understanding of the human genome make variant classification an extremely difficult job. However, with each passing day our knowledge increases, and the tools to help us become increasingly more efficient. Let’s pick up where we left off in our first article about variants. After checking Ensemble to…

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ChIP-seq Workflows Run Best with Automated DNA Size Selection

Chromatin immunoprecipitation sequencing, better known as ChIP-seq, is a massively parallel approach for understanding the interactions between proteins and DNA. This is especially important for determining the activity of transcription factors, which is why it’s frequently used to learn about the complicated series of biological steps leading to cancer. It’s also key to many epigenetic…

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Generating High-Quality Genome Assemblies from Metagenomic Sequencing

The decreasing costs in genomic sequencing over the past decade have inspired researchers to apply shotgun next-generation sequencing to entire microbial communities. While the reads generated typically cannot be assembled cleanly into individual genomes, there is often enough information produced to identify most microbes present in the population. However, this approach lacks sufficient resolution to…

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Get to Know Your Reference Genome (GRCh37 vs GRCh38)

Whether your experiment relies upon a reference-based genome assembly or mapping reads to a reference genome to identify variants, you need to choose a human reference genome assembly. But wait! You go to the FTP site of NCBI’s refseq and click on the Homo sapiens folder. There you are presented with two choices. Which one…

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CRISPR-Inspired Method Targets Large, Repetitive DNA Elements

Target capture through PCR has been a mainstay in genomics for years, but scientists working on especially repetitive, poorly characterized, or rapidly evolving regions continue to struggle to fish out those stretches of DNA for further study. However, whole genome sequencing, the only other alternative for these regions, can force researchers to pay for much…

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De Novo DNA Sequencing and the Special k-mer

The technology for DNA sequencing was developed back in 1977 thanks to Frederick Sanger. It took a bit longer before it was possible to sequence a complete genome. This is because we needed an appropriate mathematical model and massive computational power to assemble millions or billions of small reads to a larger complete genome. Today’s…

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